Gene transfer is now widely recognized as a powerful tool for analysis of biological events and disease processes at both the cellular and molecular level (Murray, E. J., ed. Methods in Molecular Biology, Vol. 7, Humana Press Inc., Clifton, N.J., (1991); Kriegler, M., A Laboratory Manual, W. H. Freeman and Co., New York (1990)). More recently, the application of gene therapy for the treatment of human diseases, either inherited (e.g., ADA deficiency) or acquired (e.g., cancer or infectious disease), has received considerable attention (Mulligan, R. C., Science 260: 926-932 (1993), Tolstoshev, P., Annu. Rev. Pharmacol. Toxicol. 32: 573-596 (1993), Miller, A. D., Nature 357: 455-460 (1992), Anderson, W. F., Science 256: 808-813 (1992), and references therein). With the advent of improved gene transfer techniques and the identification of an ever expanding library of "defective gene"-related diseases, gene therapy has rapidly evolved from a treatment theory to a practical reality.
Traditionally, gene therapy has been defined as "a procedure in which an exogenous gene is introduced into the cells of a patient in order to correct an inborn genetic error" (Blaese, R. M., Clin. Immunol. Immunopath. 61: S47-S55 (1991)). Although more than 4500 human diseases are currently classified as genetic, (Roemer, K. and Friedmann, T., Eur. J. Biochem. 208: 211-225 (1992) and references cited therein), specific mutations in the human genome have been identified for relatively few of these diseases. Until recently, these rare genetic diseases represented the exclusive targets of gene therapy efforts. Accordingly, most of the N.I.H. approved gene therapy protocols to date have been directed toward the introduction of a functional copy of a defective gene into the somatic cells of an individual having a known inborn genetic error (Miller, A. D., Nature 357: 455-460 (1992)). Only recently, have researchers and clinicians begun to appreciate that most human cancers, certain forms of cardiovascular disease, and many degenerative diseases also have important genetic components, and for the purposes of designing novel gene therapies, should be considered a "genetic disorders" (Roemer, K. and Friedmann, T., 1992, supra.). Therefore, gene therapy has more recently been broadly defined as "the correction of a disease phenotype through the introduction of new genetic information into the affected organism" (Roemer, K. and Friedmann, T., 1992, supra.).
Two basic approaches to gene therapy have evolved: (1) ex vivo gene therapy and (2) in vivo gene therapy. In ex vivo gene therapy, cells are removed from a subject and cultured in vitro. A functional replacement gene is introduced into the cells (transfection) in vitro, the modified cells are expanded in culture, and then reimplanted in the subject. These genetically modified, reimplanted cells are reported to secrete detectable levels of the transfected gene product in situ (Miller, A. D., Blood 76: 271-278 (1990)); Selden, R. F., et al., New Eng. J. Med. 317: 1067-1076 (1987)). The development of improved retroviral gene transfer methods (transduction) has greatly facilitated the transfer into and subsequent expression of genetic material by somatic cells (Cepko, C. L., et al., Cell 37: 1053-1062 (1984). Accordingly, retrovirus-mediated gene transfer has been used in clinical trials to mark autologous cells and as a way of treating genetic disease (Rosenberg, S. A., et al., New Eng. J. Med. 323: 570-578 (1990); Anderson, W. F., Human Gene Therapy 2: 99-100 (1991)). Several ex vivo gene therapy studies in humans have already begun (reviewed in Anderson, W. F., Science 256: 808-813 (1992) and Miller A. D., Nature 357: 455-460 (1992)).
In in vivo gene therapy, target cells are not removed from the subject. Rather, the transferred gene is introduced into cells of the recipient organism in situ, that is, within the recipient. In vivo gene therapy has been examined in several animal models (reviewed in Felgner, P. L. and Rhodes, G., Nature 349: 351-352 (1991)). Several recent publications have reported the feasibility of direct gene transfer in situ into organs and tissues such as muscle (Ferry, N., et al., Proc. Natl. Acad. Sci 88: 8377-8781 (1991); Quantin, G., et al., Proc. Natl. Acad. Sci. USA 89: 2581-2584 (1992)), hematopoietic stem cells (Clapp, D. W., et al., Blood 78: 1132-1139 (1991)), the arterial wall (Nabel, E. G., et al., Science 244: 1342-1344 (1989)), the nervous system (Price, J. D., et al., Proc. Natl. Acad. Sci. 84: 156-160 (1987)), and lung (Rosenfeld, M. A., et al., Science 252: 431-434 (1991)). Direct injection of DNA into skeletal muscle (Wolff, J. A., et al., Science 247: 1465-1468 (1990)), heart muscle (Kitsis, R. N., et al., Proc. Natl. Acad. Sci. USA 88: 4138-4142 (1991)) and injection of DNA-lipid complexes into the vasculature (Lim, C. S., et al., Circulation 83: 2007-2011 (1991); Ledere, G. D., et al., J. Clin. Invest. 90: 936-944 (1992); Chapman, G. D., et al., Circ. Res. 71: 27-33 (1992)) also has been reported to yield a detectable expression level of the inserted gene product(s) in vivo.
It was initially assumed that hematopoietic stem cells would be the primary target cell type used for ex vivo human gene therapy (see e.g., Wilson, J. M., et al., Proc. Natl. Acad. Sci 85: 3014-3018 (1988)), in part, because of the large number of genetic diseases associated with differentiated stem cell lineages (Miller, D., Nature 357: 455-460 (1992)). However, because of problems inherent to hematopoietic stem cell transfection (e.g., inefficient transgene expression) (Miller, A. D., Blood 76: 271-278 (1990)), more recent gene therapy efforts have been aimed at the identification of alternative cell types for transformation. These include: keratinocytes (Morgan, J. R., et al., Science 237: 1476-1479 (1987)), fibroblasts (Palmer, T. D., et al., Proc. Natl. Acad. Sci. 88: 1330-1334 (1991); Garver Jr., R. I., et al., Science 237: 762-764 (1987); International Patent Application PCT/US92/01890, having publication number WO 92/15676), lymphocytes (Reimann, J. K., et al., J. Immunol. Methods 89: 93-101 (1986)), myoblasts (Barr, E. and Leiden, J. M., Science 254: 1507-1509 (1991); Dai, Y. et al., PNAS 89: 10892-10895 (1992); Roman, M., et al., Somatic Cell and Molecular Genetics 18: 247-258 (1992)), smooth muscle cells (Lynch, C. M. et al., Proc. Natl. Acad. Sci. USA 89: 1138-1142 (1992), and endothelial cells (Nabel, E. G., et al., Science 244: 1342-1344 (1989), International Patent Application PCT/US89/05575, having publication number WO 90/06997), the contents of which references and patent/patent applications are incorporated herein by reference.
Despite the wide range of cell types tested, a satisfactory target cell for human gene therapy has not yet been identified. The inadequacies of the above-identified cell types include: (1) inefficient (Williams, et al., 1984; Joyner, et al., 1985) or transient (Mulligan, R. C., Science 260: 926-932 (1993)) expression of the inserted gene; (2) potential tumorigenicity of the implanted transduced cell (Selden, et al., 1987; Garver, et al., 1987b); (3) rejection of the implanted genetically modified cell in the absence of harsh immunosuppressive therapy (Selden, et al., 1987); (4) necrosis following subcutaneous injection of cells (Bell, et al., 1983); (5) limited dissemination of the inserted gene product from the site of transduced cell implantation (Morgan, et al., 1987) (see also WO 92/15676); and (6) limitations in the amount of therapeutic agent delivered in situ.
The delivery of a therapeutically effective dose of a therapeutic agent in situ depends on both the efficiency of transfection (or transduction), as well as the number of target cells. Thus, despite the potentially high efficiency of transduction using retroviral vectors, many of the above-described cell types are not satisfactory target cells for in vivo gene therapy because of the relatively small numbers of cells available for transduction in situ. Similarly, many of these cell types are not satisfactory for ex vivo gene therapy because of the limited area available in situ for receiving (e.g., by implantation) the genetically modified cells or because of inherent difficulties in accessing a particular anatomical location for implantation of the genetically modified cells.
Endothelial cell-based gene therapy, in particular, is limited by the relatively small area available in situ for receiving genetically modified endothelial cells. Typically, ex vivo gene therapy using endothelial cells requires that a relatively small portion of a blood vessel be sectioned-off to eliminate blood flow through the vessel before removing cells from, or introducing cells to, the blood vessel (Nabel, E. G., et al., Science 244: 1342-1344 (1989); Lim, C. S. et al., Circulation 83: 2007-2011 (1991); Chapman, G. D. et al., Circulation Res. 71: 27-33 (1992)). Consequently, a relatively small number of genetically modified endothelial cells can be implanted into the target vessel. As a result, the delivery of a therapeutically effective dose of therapeutic agent in situ is limited by the total number of implanted endothelial cells.